Two tropical seagrass species show differing indicators of resistance to a marine heatwave

Abstract Marine heatwaves (MHWs) are a growing threat to marine species globally, including economically and ecologically important foundation species, such as seagrasses. Seagrasses in tropical regions may already be near their thermal maxima, and, therefore, particularly susceptible to increases in temperature, such as from MHWs. Here, we conducted a 10‐day MHW experiment (control +4°C) to determine the effects of such events on the two tropical seagrasses Halophila beccarii and Halophila ovalis. We found that both species were largely resistant to the MHW, however, there were differences between the species' responses. For H. beccarii, the surface area of existing leaves was smaller under MHW conditions, yet a substantial increase in the number of new leaves under the MHW indicated its tolerance to—or even increased performance under—the MHW. While there was no direct effect of the MHW on H. ovalis, this species saw less epiphyte biomass and percentage cover on its leaves under the MHW. While a lower epiphyte cover can potentially increase the health and ecophysiological performance of the seagrass, the change of epiphytes can lead to bottom‐up trophic implications via the influence on mesograzer feeding. Together, the results of this study demonstrate the species‐specific responses of seagrasses of the same genus to a warming event. With the current global decline of seagrasses, our results are encouraging for these important habitat formers as we show that anomalous warming events may not necessarily lead to ecosystem collapse.

Climate change and other anthropogenic impacts can drive large-scale effects on seagrasses, with these meadows disappearing at a rapid rate (Orth et al., 2006;Short & Neckles, 1999;Waycott et al., 2009). Over recent decades loss of seagrass has been reported to be occurring globally at a rate of approximately 110 km 2 year −1 , making them one of the most threatened ecosystems in the world (Waycott et al., 2009). Specifically, seagrass loss has been recorded in the Americas , the Mediterranean (Marbà et al., 2014), Asia (Unsworth et al., 2018) and Australia (Kendrick et al., 2019). Additionally, further loss, distribution shifts and altered ecophysiological performance are expected in the future as anthropogenic impacts increase (Chefaoui et al., 2018;Nguyen et al., 2000;Repolho et al., 2017).
One climate change factor that can have, and is already having, a prominent effect on seagrasses is marine heatwaves Smith et al., 2023). Marine heatwaves (MHWs) are defined as five or more days of anomalous sea surface temperature above the regional 90th percentile (Hobday et al., 2016). These MHW events are increasing in intensity, frequency and duration and are predicted to continue doing so in the future . MHWs can have important ecological effects and have been shown to degrade seagrass in temperate regions (Arias-Ortiz et al., 2018;Marbà & Duarte, 2010;Serrano et al., 2021;Strydom et al., 2020;Thomson et al., 2015). Tropical seagrass species have important interactions with mangroves and corals (Green & Short, 2003) and while seagrass studies in the tropics were previously lacking (Unsworth et al., 2018), there has been an increase in research considering their responses to anomalous warming events (Rasmusson et al., 2021;Strydom et al., 2020;Szitenberg et al., 2022;Thomson et al., 2015). It is important to study seagrasses in these regions given that the bulk of extant seagrass meadows is found on the coastline of developing tropical countries (Duarte, 2002), which are experiencing the greatest rate of environmental degradation and are projected to continue to do so in the future.
The loss of seagrass meadows can have repercussions for many trophic levels and on the whole ecosystem functioning.
That is, seagrasses are an important food source for macro herbivores (Bjorndal, 1980;Marsh et al., 1982) and mesograzers (Ebrahim et al., 2014;Klumpp et al., 1992), as well as a blue carbon sink (Gullström et al., 2018). In tropical systems extreme climatic events can have severe effects on traits (e.g. survival, abundance) of habitat-forming species including seagrasses, with prolonged impacts for slow-growing species than their faster-growing counterparts (Babcock et al., 2019).  (Short et al., 2007). For the two species used in this study, H. ovalis has a wide subtropical and tropical distribution, ranging from the west coast of Africa to South Asia, South-East Asia and Australia.
H. beccarii also has a wide, yet patchy, distribution in the tropical Indo-Pacific (Green & Short, 2003). In Hong Kong, these species are found on the low intertidal and shallow subtidal on mudflats, and are associated with mangroves and also with seaweed in spring (Morton & Morton, 1983, personal observation    , the temperature treatments in the experiment were a control (the average spring SST for Hong Kong which is the season when this experiment was done-23°C) and a 'severe' category MHW treatment  of +4°C above control temperature (to represent a likely intensity of near-future MHWs-27°C). Halophila ovalis in Tung Chung Bay covered an area of approximately 15,928 m 2 and the density was 300-400 shoots per 25 cm 2 . The seagrass was predominantly distributed in a large meadow, particularly toward the west of the site, however, there were also small patches of H. ovalis distributed irregularly throughout the mudflat. Seagrass and sediment adjacent to the meadows were collected using a trowel and placed into cool boxes. Spot measurements of temperature, pH and salinity were also taken during collection. For Tung Chung, the water temperature was 22.4°C, pH was 7.96 and salinity was 27 ppt, whereas at Pak Nai, water temperature, pH and salinity were 23.7°C, 8.24 and 24 ppt, respectively. Seagrass and sediment were transported to the Marine Science Laboratory at The Chinese University of Hong Kong, and the seagrass was placed in outdoor tanks and allowed to acclimate to ambient tank conditions for 2 days (as in Bass et al., 2023;Noisette & Hurd, 2018), with natural 12:12 h day/night light conditions. The weather was mostly overcast during the experiment and daytime irradiance levels outside varied between 250 and 1850 μmol m −2 s −1 depending on the weather conditions (MQ-510

| Seagrass collection
Full Spectrum Underwater Spectrum Meter; Apogee Instruments, Inc.). Following the acclimation period, individual ramets (seagrass shoots, attached to the rhizome and roots) were sorted, had initial measurements taken, were then planted into experimental tanks with the collected sediment (which was filtered using a metal sieve mesh size 4 mm-to remove biological material) with ambient air pumped into each tank and exposed to treatment temperatures.
To establish and maintain temperature treatments, seawater from the adjacent Tolo Channel was pumped into header tanks (one for each temperature treatment), which was connected to a chiller (HC-1000B Chiller, Hailea®, China) for the control temperature treatment, or had water heaters inside (D-839-1000, Up-aqua™, Taiwan) for the MHW treatment. This water was then pumped into each experimental tank, after which the water was able to flow out of the experimental tanks in a continuous flow-through system (see Figure S1 for experimental setup schematic). Experimental temperatures were measured using temperature data loggers (iButton™, United Kingdom) placed into the experimental tanks (4-5 loggers per treatment) which were set to record every 10 min.  Duarte, 2001) in order to identify ramet and correct orientation for measurements at the end of the experiment. Ramets were then photographed before planting the ramets into the filtered sediment. Initial measurements were later recorded from these photographs, including rhizome length (horizontal and vertical rhizomes included), root length and leaf surface area using Fiji extension of Image J (Schindelin et al., 2012). Analysis of these initial measurements revealed that seagrasses of the same species had the same starting measurements across treatments, and, therefore, there was no initial bias.
After the 10-day exposure to control or MHW water temperature, the seagrass was taken from their experimental tanks and rinsed gently with seawater to remove sediment. Ramets were identified and those which had completely disintegrated or were unidentifiable were categorised as 'dead'. As mortality was low for both species, this response variable was excluded from formal statistical analysis.
For those ramets that had survived, photographs were again taken and rhizome length, root length and the presence (and surface area) or loss of the original leaves were measured and analysed in ImageJ.
Epiphyte percentage area was also recorded for the original leaves.
For the new growth, that is, rhizome, roots, shoots and leaves that were not present at the start of the experiment, length measurements were taken of new rhizome growth, new root growth, the number of new leaves grown and also surface area measurements of the largest new leaf.
After taking the photographs for the above measurements, seagrass ramets were divided into sections for dry-weight biomass quantification. These sections included epiphyte biomass (from three of the old leaves present at the initial measurement), leaf biomass (of the three old leaves and then of all leaves together), rhizome biomass and total root biomass. The sections of each ramet were placed in foil (pre-weighed) before drying at 60°C for 2 days. Once measured, ramet total dry weight biomass was calculated by totalling the amounts of each individual section (with the exception of epiphyte biomass).

| Statistical analyses
Measurements were then analysed in the statistical software R (version 4.2.0). The two species were analysed separately due to their inherent differences in morphology and growth strategies.
For each response variable and each species, a mixed effects model was run with fixed factor 'treatment' and random effect of 'tank number' (i.e. each tank was treated as a replicate). A Levene's test was carried out for each species and response variable for test for homogeneity of data (Brown & Forsythe, 1974). Data were then transformed if needed (log or square root) to conform to normalisation and then the mixed effects model was performed using the package 'lme4' (Bates et al., 2015). Analysis of deviance using the Anova function was performed using the package 'car' (Fox & Weisberg, 2011) and p-values of <.05 were used for significance.
For zero-inflated data, that is, epiphyte biomass for H. beccarii, a zero-inflated model was fitted using the package 'glmmTMB' with a 'beta' transformation and a p-value extracted using ANOVA, type III. on length or area for any of the seagrass segments (Figure 2b,d,f; In terms of changes in the properties of leaves present, there was no difference in the size of new leaves grown on each ramet for either species in the control or MHW treatment (Figure 3a;

| RE SULTS
Tables S1 and S2). The number of leaves at the end of the experiment compared to the start was, however, substantially boosted under MHW conditions for H. beccarii (averages of 14.20 leaves in MHW conditions compared to 6.13 under control conditions; Figure 3b; Table S1; F = 7.89, p < .05), which was associated with a greater production of new leaves (averages of 21.7 under MHW vs. 14.0 new leaves at control treatment; Figure 3c, Table S1, F = 6.14, p < .005).
For H. ovalis, there was no difference in either change in leaf number or number of new leaves between the control and MHW treatment (Figure 3b,c; Table S2). There was also no difference in the number of leaves lost during the experiment between treatments for either species (Figure 3d; Tables S1 and S2).

| DISCUSS ION
With climate change, there is an increase in the number, intensity and duration of MHW events (Frölicher et al., 2018). As a result, there is recorded global loss of seagrass, particularly loss of density, as well as subsequent changes to community structure . In areas of East and South-East Asia, seagrass loss has been recorded in Vietnam (Luong et al., 2012), however, there are relatively few recordings of MHW effects on seagrasses in these tropical regions compared to records made in temperate regions (Bennett et al., 2022;Deguette et al., 2022;Fraser et al., 2014). The present study shows significant resistance of two seagrass species, Seagrasses, including Halophila spp., typically have high growth rates and fecundity which allow them to respond rapidly to environmental perturbations (Marbà & Duarte, 1998;O'Brien et al., 2018;Rasheed et al., 2008). Halophila ovalis has an approximate plastochron interval of 4.4 days for each pair of leaves (Erftemeijer & Stapel, 1999;Vermaat et al., 1995), while for H. baccarii plastochron interval has not been defined but new shoots can be formed within 2-3 days (Le Xuan et al., 2022). In this study, the production of new leaves for H. ovalis was similar under the different temperature treatments and corresponded to the rates of the known plastochron interval for this species. In contrast, H. beccarii increased its leaf turnover rate by boosting the number of new leaves grown under the MHW condition compared to ramets grown under the control condition. Similar enhancement is also seen in other seagrass species under simulated warming, such as increased metabolism and leaf production rate (Egea et al., 2019;Liu et al., 2022). The MHW condition in this study was 27°C, which likely falls within the thermal tolerance range of these species: while H. beccarii's thermal optimal range has not been formally recorded, that of H. ovalis is recorded to be between 25 and 30°C (Ralph, 1998), and subtropical and tropical seagrasses typically have a thermal optimal range up to 35°C (Campbell et al., 2006;Ralph, 1998). For the genus Halophila and other small,

F I G U R E 4 Epiphyte properties of Halophila beccarii and
Halophila ovalis leaves exposed to marine heatwaves (control vs. heatwave) in terms of: (a) epiphyte biomass and (b) per cent cover of epiphytes. Asterisks represent level of significance (*p < .05, **p < .01). fast-growing species, there is a characteristic tendency for higher above-ground biomass production in comparison to below-ground (Duarte & Chiscano, 1999). As the above-ground seagrass sections can contribute to photosynthesis and higher productivity, increasing below-ground biomass may become a respiratory burden under increased temperature (Lee et al., 2007) (Ranjitham et al., 2008).
The results from this study also indicate that increased temperature from a MHW can affect the root length of H. beccarii, which was lower under the MHW treatment. Reduced root length can be a self-thinning mechanism of the seagrass in response to high seagrass density (Jiang et al., 2020). Although the seagrass in the experimental tanks was not overcrowded, there was higher above-ground biomass of H. beccarii under the MHW treatment by the end of the experiment, which may have generated this response in root length. There are various negative consequences of restricted root length, such as decreased resistance to hydrodynamic exposure and reduced capacity to absorb nutrients (Jiang et al., 2020). However, lowered root length can also shorten the path length for oxygen supply (Hovey et al., 2012;Jiang et al., 2019). This change can be beneficial to the seagrass, particularly as the oxygen requirement for respiration is higher under elevated temperatures (Rasmusson et al., 2020). Therefore, in response to MHWs, shorter root length may be advantageous and facilitate increased respiration rates.
Changes in epiphyte cover can have important implications for both the seagrass it occurs on and the species which consume it.
Here, we identified that H. ovalis had lower biomass and percentage cover of epiphytes on its leaves under the MHW treatment. Lower epiphyte biomass can be beneficial to the seagrass as it improves access to light, consequently leading to better health of the seagrass (Brodersen et al., 2015) and increasing seagrass productivity . In contrast, the epiphytes that grow on the seagrass leaves are also a considerable food source for mesograzers. For example, several gastropod species have been recorded grazing on the epiphytes of H. ovalis (Fong et al., 2018). Seagrassepiphyte-grazer relationships have been studied rigorously in the past 40 years (Duffy et al., 2001;van Montfrans et al., 1984), and show that loss of epiphytes on seagrass leaves can affect the grazing species and translate to changes on many trophic levels (Bendell, 2009 While the results of this study demonstrate the encouraging positive and neutral responses of the two species to short MHWs, longer MHWs events can increase the severity of deleterious physiological responses from seagrasses (Olsen et al., 2012;Pruckner et al., 2022), particularly for events when the ambient condition is warmer (i.e. summer MHWs; Bass et al., 2023). Moreover, stressors co-occurring with MHWs can lead to more deleterious effects, such as changes in nutrient levels and turbidity, which can have much larger repercussions on the seagrass ecosystems (Kendrick et al., 2019;Orth et al., 2006). In large coastal cities, such as Hong Kong, land reclamation, pollution and harmful algal blooms threaten coastal habitat loss and fragmentation (Lai et al., 2016). These disturbances, along with extreme climatic events, such as tropical cyclones (Fong, 2000) and MHWs of increased severity and duration have to potential to compound and exacerbate the currently observed patterns of global seagrass loss.

| CON CLUS ION
This study indicates that short MHWs in springtime pose no significant additional threat to the survival or growth of these two seagrass species. Importantly, their differing responses, and the re-